The field of this invention is gene expression. More particularly, this invention pertains to a process for increasing the circulating levels of gene products over an extended period of time.
A large number of inherited and acquired serum protein deficiencies including hemophilia A, diabetes mellitus and the erythropoietin-responsive anemias are currently treated by repeated intravenous or subcutaneous injections of purified or recombinant proteins. Although largely effective, such therapies are both expensive and inconvenient. Moreover, in diseases such as hemophilia A, there is not sufficient recombinant protein available to allow a comprehensive program of prophylactic therapy. Given these problems, there has been considerable interest in developing novel gene-based therapies for such serum protein deficiencies. An initial series of studies demonstrated that skeletal myoblasts genetically modified in vitro could be reimplanted by intramuscular injection and would subsequently produce stable, physiological levels of recombinant proteins in the systemic circulation of adult immunocompetent mice. Subsequently, several groups have demonstrated the stable production of recombinant serum proteins following a single intramuscular (IM) injection of replication-defective adenovirus (RDAd) vectors. Despite these initial successes, both myoblast transplantation and IM injection of RDAd vectors have thus far been associated with problems that may preclude their widespread clinical application.
The studies reported to date have all been done on rodents such as mice. Those data may not reflect and may not be predictive of results in larger animals such as primates. It is well known in the art, for example, that physiological or therapeutic doses observed in rodents are not necessarily predictive of effective doses in larger mammals. Still further, the amount of vector needed in large mammals may preclude their utility. For example, the mass of vector needed in primates may be so large that their injection results in either adverse reactions to the injection (e.g., anaphylactic shock), generation of an immune response or secondary infection resulting from the use of large numbers of viral particles. Still further, the data from previous reports do not address the question of whether there is any correlation between the amount or dose of transforming vectors and increases in the levels of gene products. There continues to be a need in the art, therefore, for processes for increasing the circulating levels of gene products in large mammals such as primates.
The present invention provides a process of increasing the circulating levels of a gene product in the blood stream of a mammal for a period of time greater than about 30 days. The process includes the step of transforming muscle cells of the mammal with a polynucleotide that encodes the gene product, wherein the expression vector drives long-term expression of the polynucleotide.
A preferred mammal is an animal used for food such as a cow, domesticated animals such as dogs and cats and primates. A preferred primate is a human. A process of the present invention can be used to increase circulating levels of any gene product. Exemplary such gene products are RNA molecules, single-stranded DNA molecules and polypeptides. Polypeptides are particularly preferred. Especially preferred polypeptides are polypeptide hormones such as growth hormone and erythropoietin.
A process of the present invention can use any muscle such as smooth muscle, cardiac muscle and skeletal muscle. Cardiac and skeletal muscle are preferred. The use of skeletal muscle is most preferred.
Any suitable expression vector can be used in the present process. Exemplary and preferred such vectors are plasmids and replication defective adenoviral vectors. The muscle cells can be transformed either in vivo or ex vivo. When transformed in vivo, the expression vector is directly injected into a muscle mass of the mammal. When transformed ex vivo, muscle cells are removed from the mammal, transformed ex vivo and the transformed muscle cells reimplanted into the mammal.
A process of the present invention is useful for increasing the circulating levels of gene products over an extended period time. Using a process of this invention, those levels can be increased for periods of time ranging from greater than about 60, 90 or 120 days and even for as long as one year.
The safety and efficacy of IM injection of adenoviral vectors encoding Epo in both mice and non-human primates has been determined. In an initial series of experiments, the relationship between the dose of vector injected and the corresponding elevations in serum Epo levels and hematocrits in both species were studied. The results demonstrated that there is a threshold dose in both mice and monkeys (approximately 2.5-8xc3x97107 pfu/gm body weight) that is required to obtain long-term Epo expression and polycythemia. A single IM injection of mice with 109 pfu of vector resulted in elevations in hematocrits from control values of 49% to treated values of 81% which were stable for more than one year. Similarly, a single IM injection of a monkey with 4xc3x971011 pfu of an adenoviral vector encoding simian Epo (AdsEpo) resulted in elevations of hematocrits from control levels of 40% to treated levels of =70% which were stable for 84 days. IM injection of monkeys with vector was determined to be safe in that no abnormalities in chest X-rays, serum chemistries, hematologic or clotting profiles (except for elevated hematocrits) or organ pathology were seen during the 84 day time course of the experiment.
The present invention provides a process of increasing the circulating level of a gene product in the blood stream of a mammal for a period of time greater than about 30 days. The process includes the step of transforming muscle cells of the mammal with a polynucleotide that encodes the gene product, wherein the expression vector drives long-term expression of the polynucleotide.
As is well known in the art, gene products include polynucleotides such as DNA and RNA and polypeptides. As is also well known in the art, those gene products can be secreted from the cells where they are made into the extracellular fluid compartment of the organism. From there, those products diffuse into the blood. The process of the present invention can be used to increase the levels of those gene products in the blood over an extended period of time. A process of this invention is particularly useful in increasing the levels of polypeptide gene products. Preferably, the polypeptide is secretory product of a cell. Exemplary such products are cytokines, colony stimulating factors, nerve growth factors and the like. Exemplary and preferred such secretory products are polypeptide or protein hormones. Such hormones are well known in the art. Exemplary polypeptide hormones are insulin, glucagon, renin, parathyroid hormone, growth hormone, erythropoietin and the like.
A process of the present invention can be used to increase the circulating level of a gene product in any mammal. The process is particularly useful in large mammals such as domestic pets, those used for food production and primates. Exemplary large mammals are dogs, cats, horses, cows, sheep, deer and pig. Exemplary primates are monkeys, apes and humans. The use of the present process in humans is particularly preferred.
The present invention discloses that increased circulating levels of gene products can be realized by transforming muscles cells of the mammal with a polynucleotide that encodes that gene product. As is well known in the art, mammals contain three types of muscle cells: smooth muscle, cardiac muscle and skeletal muscle. Any one of these muscle types can be used in a present process. Because of the accessibility of large masses of cardiac and skeletal muscle, use of these muscle types is preferred. In an especially preferred embodiment, a process of this invention uses skeletal muscle.
As used herein, the phrase xe2x80x9cexpression vectorxe2x80x9d means any vehicle for delivering an encoding polynucleotide to a cell such that the polynucleotide is expressed and a gene product is formed. Expression vectors are well known in the art. As is also well known in the art, particular vectors are especially suitable for transforming mammalian cells. For use in the present invention, plasmids and viral vectors are preferred. Exemplary viral vectors include retroviral vectors and adenoviral vectors. The vectors, especially the adenoviral vectors, are made replication defective using standard procedures well known in the art. The choice of a particular vector depends inter alia on the nature of the gene product to be produced.
Replication-defective adenoviruses represent an efficient and safe method of in vivo gene transfer. These vectors can be prepared at high titer (up to 1011 pfu/ml) and infect many replicating and non-replicating cell types in vivo. Adenoviruses are common and relatively benign human pathogens that have not been associated with persistent infections or neoplasias in humans. Wild-type adenoviruses have been used previously for human vaccination. As disclosed herein, a single IM injection of a replication-defective adenovirus encoding hEpo can be used to produce dose-dependent elevations in serum Epo levels and hematocrits which were stable over the 120 day time course of these experiments. The injected adenovirus remains localized at the site of administration and does not cause muscle pathology. Taken together, these results show that IM injection of replication-defective adenoviruses is useful for the treatment of a number of acquired and inherited human serum protein deficiencies.
Thus, in one embodiment, the expression vector is a replication defective adenoviral vector. A single IM injection of immunocompetent mice with 109 to 3xc3x97109 plaque forming units (pfu) of an E1-and E3-deleted replication-defective adenovirus vector encoding murine Epo (AdmEpo) resulted in elevations of serum Epo levels and hematocrits from control values of approximately 45% to treated values of approximately 80% which were stable for at least 112 days (Tripathy et al., Nature Med. 2, 545-550, 1996).
As disclosed herein, an adenoviral vector, RdAd, encoding self Epo can be used to produce sustained and significant elevations in hematocrits in both mice and non-human primates. Unlike previous observations in immunocompromised SCID animals, the persistence of transgene expression in immunocompetent animals appears to be critically dependent upon the dose of virus administered, with a threshold dose of approximately 2.5-8xc3x97107 pfu/gm in both mice and monkeys required to obtain persistent transgene expression. From a safety standpoint, there was no evidence of pulmonary or hepatic toxicity and there was no demonstrable long term organ pathology in monkeys injected once IM with 4xc3x971010-4xc3x971011 pfu of AdsEpo. Taken together, these results show that IM injection of RdAd encoding human Epo can be used to safely and effectively treat patients with Epo-responsive anemias.
Previous studies in immunocompromised animals demonstrated that IM injection of SCID mice with as little as 107 pfu of Epo-encoding RdAd resulted in stable elevations in hematocrits. Increasing the viral dose to 108 or 109 pfu produced further increases in both serum Epo levels and hematocrits (Tripathy et al., Proc. Natl. Acad. Sci. USA, 91, 11557-11561, 1994). Thus, in SCID animals there appeared to be a simple linear relationship between the dose of virus administered and the resulting levels of Epo in the serum. The data disclosed herein in immunocompetent mice and monkeys clearly demonstrate a more complex relationship between viral dose and hematocrit. IM injection of immunocompetent mice and monkeys with low doses of virus resulted in only transient increases in hematocrit, whereas injection with higher doses of virus led to sustained elevations in serum Epo levels and hematocrits. Moreover, there appeared to be a threshold dose (2.5-8xc3x97107 pfu/gm) in both species that was required for persistent transgene expression.
The observed differences between the SCID and immunocompetent animals clearly implicated the immune system as the critical determinant of these different dose-response relationships. However, there are several possible alternative mechanisms that might explain these differences. First, it is possible that the immune system eliminates a significant fraction of the Epo-expressing myocytes, independent of the dose of virus administered. In this case, initial infection of a relatively large number of myocytes might be required to end up with a sufficient number of Epo-producing cells to produce physiologic elevations in serum Epo levels. Such a model is supported by our measurements of serum Epo levels following IM injection of immunocompetent mice with AdmEpo. Peak serum Epo levels observed 1 week following viral infection subsequently declined by approximately 70% by 4 weeks after injection and then stabilized. In the animals injected with 108 pfu of AdmEpo this decline resulted in serum Epo levels that fell to or below endogenous circulating Epo levels and therefore did not result in a polycythemia. In contrast, in the animals injected with 109 pfu of AdmEpo, this decline still resulted in serum Epo levels that were approximately 10-fold above pre-injection levels, thereby leading to significant elevations in hematocrit.
Alternatively, it is possible that the type of immune response elicited by IM injection of the AdEpo vectors is critically dependent upon the dose of vector administered. It has recently been shown that exposure of mice to high doses of antigen leads predominantly to Th2 responses, whereas administration of lower doses of antigen leads to Th1. Therefore, it is possible that Th1 responses to the low dose of AdmEpo in the animals receiving 4xc3x971010 pfu of virus led to CTL-mediated elimination of virus infected cells, whereas Th2 responses to high doses of vector allowed persistence of larger numbers of Epo-producing myocytes in the animals that received 4xc3x971011 pfu of virus. These two mechanisms are, of course, not mutually exclusive.
The dose of virus (in pfu/gm) required to produce sustained transgene expression in mice and monkeys was quite similar. This finding indicates that it is possible to predict human doses based upon our rodent and primate data. From the mouse data, a single injection of 109 pfu of AdmEpo resulted in a sustained 30 point elevation in hematocrit (from approximately 50% to 80%). Thus, it requires approximately 1.33xc3x97106 pfu/gm body weight to produce a 1 point elevation in hematocrit. For example, to produce a 15 point increase in hematocrit in a 70 kg human with Epo-responsive anemia (eg., to increase the hematocrit from 23% to 38%) it would require a single IM injection of (1.33xc3x97106 pfu/gm)xc3x9715 (% increase in hematocrit)xc3x9770,000 gm=1.4xc3x971012 pfu of AdhEpo. A previously reported adenoviral vector, AdhEpo, which is identical to AdmEpo and AdsEpo except that it contains the human Epo cDNA, has been shown to drive the expression and secretion of high levels of human Epo following both in vitro infection of myocytes and IM injection of SCID mice (Tripathy et al.,. Proc. Natl. Acad. Sci. USA, 91, 11557-11561, 1994).
The lack of toxicity observed in Cynomologus monkeys following IM injection of 4xc3x971011 pfu of AdsEpo, a dose sufficient to produce significant elevations in hematocrits, also augurs well for the safety of human gene therapy using IM injection of Epo-encoding vectors. This relative lack of toxicity as compared to previous experiments involving intravenous or inhaled routes of administration may reflect the fact that relatively little vector infects the liver or lung following IM administration. In addition, because previous experiments have demonstrated that immune responses to foreign transgenes play an important role in the inflammatory responses to RDAd-infected cells (Tripathy et al., Nature Med. 2, 545-550, 1996), the use of a self-transgene (to which the animal is tolerant) may have significantly reduced immune responses to the vector-infected cells in these animals. The present finding of excessive polycythemia in the monkey injected with 4xc3x971011 pfu of AdsEpo suggested that it will be essential to begin human trials with low doses of vector in order to carefully assess dose-response relationships in humans. However, the fact that injected muscle can be removed to terminate therapy adds a relative safety factor to therapies involving IM as opposed to systemic or pulmonary administration of RdAd. The use of such vectors will significantly increase the safety of adenovirus-mediated gene therapy for Epo-responsive anemias. Finally, the finding of significant levels of anti-adenoviral antibodies in mice and monkeys following a single IM injection of RdAD will likely make readministration of the vector difficult or impossible. Indeed, recent studies in mice have demonstrated that it is not possible to readminister AdsEpo to mice even 9 months after an initial IM injection. Modifications of the vector or transient immunosuppresive regiments will therefore likely be necessary to obviate this problem.
In another embodiment, the expression vector is a plasmid. The IM injection of plasmid DNA has a number of distinct advantages as compared to the use of RDAd vectors. First, plasmid DNA vectors are easier to construct, can accept large cDNA inserts, and can be prepared as pure chemical solutions without the risk of contamination with wild-type infectious particles. In addition, IM injection of adult immunocompetent animals with RDAd has been associated with immune responses that eliminate virus infected cells in 14-28 days, thereby producing only transient recombinant gene expression in vivo. Of equal importance, previous infection with wild-type adenovirus results in a neutralizing antibody response which may preclude administration of an RDAd vector. In contrast, the present disclosure demonstrates long-term Epo expression following a single IM injection of plasmid DNA even in adult immunocompetent animals. Moreover, because there were no detectable antibodies against mEpo in the sera of mice 90 days after injection with pVRmEpo, it is possible to readminister plasmid DNA by IM injection if repeated therapy or dose escalation is required.
The present invention discloses the construction and characterization of a novel plasmid vector that produces high level expression and secretion of erythropoietin (Epo) following IM injection into adult immunocompetent mice. A single IM injection of as little as 10 mg of this plasmid produced physiologically significant levels of mEpo in the systemic circulation of adult immunocompetent mice and resulted in significant elevations in hematocrits that were stable for at least 90 days. The injected plasmid DNA remained localized at the site of injection and the amount of Epo production (as reflected by the elevated hematocrits) was proportional to the dose of plasmid DNA injected. Thus, IM injection of plasmid DNA represents a feasible approach to the treatment of serum protein deficiencies.
As shown in detail hereinafter in the Examples, IM injection of as little as 10 mg of pVRmEpo (a novel plasmid expression vector, pVRmEpo, which directs high level production and secretion of mEpo from skeletal myocytes in vitro) into adult immunocompetent mice resulted in dose-dependent elevations in hematocrits that remained stable for at least 90 days. The increased hematocrits observed in the pVRmEpo-injected mice reflected persistent production of mEpo from the injected muscle and secretion of this protein into the systemic circulation. Finally, the injected DNA remained predominantly localized to muscle at the site of injection. The data disclosed herein represent the first demonstration of the delivery of physiologically significant levels of recombinant protein to the systemic circulation following the IM injection of a plasmid DNA expression vector.
The expression vector drives expression of the polynucleotide that encodes the particular gene product. Thus, the vector needs to contain those expression elements necessary for expression. For example, a polynucleotide of an expression vector of the present invention is preferably operatively associated or linked with an enhancer-promoter. A promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins. That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes. As used herein, the term xe2x80x9cpromoterxe2x80x9d includes what is referred to in the art as an upstream promoter region or a promoter of a generalized RNA polymerase transcription unit.
Another type of transcription regulatory sequence element is an enhancer. An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. Unlike a promoter, an enhancer can function when located at variable distances from a transcription start site so long as the promoter is present.
As used herein, the phrase xe2x80x9cenhancer-promoterxe2x80x9d means a composite unit that contains both enhancer and promoter elements. An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product. As used herein, the phrase xe2x80x9coperatively linkedxe2x80x9d or its grammatical equivalent means that a regulatory sequence element (e.g. an enhancer-promoter or transcription terminating region) is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter. Means for operatively linking an enhancer-promoter to a coding sequence are well known in the art.
An enhancer-promoter used in an expression vector of the present invention can be any enhancer-promoter that drives expression in a host cell. By employing an enhancer-promoter with well known properties, the level of expression can be optimized. For example, selection of an enhancer-promoter that is active in specific cells (e.g., muscle cells) permits tissue or cell specific expression of the desired product. Still further, selection of an enhancer-promoter that is regulated in response to a specific physiological signal can permit inducible expression.
A coding sequence of an expression vector is operatively linked to a transcription terminating region. RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA). Enhancer-promoters and transcription-terminating regions are well known in the art. The selection of a particular enhancer-promoter or transcription-terminating region will depend, as is also well known the art, on the cell to be transformed.
The muscle cells can be transformed either in vivo or ex vivo. When transformed in vivo, the expression vector can be directly injected into muscle cells of the mammal. Alternately, the vector can be delivered to the muscle cells by infusing the vector into an artery or vein that perfuses the target muscle. Means for transforming smooth, cardiac and skeletal muscle in vivo are well known in the art. In a preferred embodiment, the vectors are directly injected into cardiac or skeletal muscle.
When transformed ex vivo, muscle cells are removed from the mammal, transformed ex vivo and the transformed muscle cells reimplanted into the primate. When ex vivo procedures are used, the use of skeletal muscle is preferred.
A process of the present invention is used to increase the circulating level of a gene product. As used herein, the term xe2x80x9cincreasexe2x80x9d means to raise the circulating level above the pre-transformation level. Thus, the present process can be used to enhance levels above the normal physiological level of that gene product or can be used to correct abnormal deficiencies in the level of that product. Further, as shown hereinafter in the Examples, the circulating level of a gene product can be increased in a dose-dependent fashion. Preferably, the pre-transformation circulating levels can be increased at least 10 percent with use of the present process. Even more preferably, the circulating levels can be increased at least 20 percent, 50 percent, 100 percent or even greater. The data set forth hereinafter in the Examples also show that the physiological activity (e.g., hematocrit) of the gene product (e.g., erythropoietin) can also be increased with use of the present process.
The Examples that follow illustrate preferred embodiments of the present invention and are not limiting of the specification and claims in any way.